S. aureus is a leading cause of community-acquired and nosocomial infections, with a propensity for biofilm formation. Biofilms are heterogeneous bacterial communities that are a serious health care concern based on their chronicity and recalcitrance to antibiotic therapy. Prior to our work, the prevailing concept was that S. aureus biofilms passively evaded immune-mediated detection to establish chronic infection. However, our studies during the initial PPG funding period challenged this dogma, demonstrating that biofilms actively skew the host innate immune response towards an anti-inflammatory state that facilitates biofilm persistence. We identified a biofilm immune signature, typified by an abundance of alternatively activated M2 macrophages (Ms) and myeloid-derived suppressor cells (MDSCs), in addition to a paucity of neutrophil and T cell infiltrates. Our central P01 hypothesis is that S. aureus biofilm architecture and metabolism create a unique niche that promotes an immune suppressive environment. In Project 4, we will identify the molecular mechanisms that favor M2 M and MDSC accumulation during S. aureus biofilm infection and the role of biofilm-derived NH4+ in this process, which addresses the immune suppressive and metabolic components of the central P01 hypothesis. We will take advantage of the immune similarities between biofilms and tumors to explore the functional importance of key cytokines (i.e. IL-6, IL-12) and effector molecules (i.e. arginase) required for MDSC and M2 M action during biofilm infection. In addition, we have utilized innovative approaches to probe for factor(s) produced by S. aureus biofilms that favor M2 M polarization, revealing an important role for NH4+-generating pathways [carbamoyl phosphate synthetase (CarA) and arginine deiminase (ArcA)] that will be investigated in the renewal application. Identifying the molecular pathways whereby S. aureus biofilms promote M2 M and MDSC infiltrates will advance our understanding towards the long-term goal of targeting these populations to facilitate biofilm clearance in the clinical setting.